1. Field of the Invention
The present invention concerns an improved test composition, device, and method for the detection of bilirubin in urine.
The diagnostic value of the determination of bilirubin in urine is well known. Urine from a normal subject does not contain an appreciable amount (less than 0.05 mg/100 ml) of bilirubin. However, various diseased conditions, such as biliary obstruction and hemolytic and hepatic diseases, cause bilirubin to appear in urine at an abnormally high concentration. It is generally recognized that the presence of bilirubin in urine at a concentration above 0.05 mg/100 ml indicates an abnormal clinical status requiring the performance of more comprehensive diagnostic procedures in order to determine the specific causative disease.
It is generally recognized that essentially all bilirubin that appears in pathological urine is in a conjugated form. Bilirubin is a degradation product of the heme moiety of hemoglobin and, once formed in the blood stream, is taken up by the liver where it becomes conjugated through esterification with glucuronic acid. The resulting bilirubin glucuronides are extremely water soluble, in contrast to free bilirubin which is highly insoluble in water The conjugated bilirubins are thus able freely to pass from the liver to the kidneys where under normal clinical conditions, essentially all conjugated bilirubin is converted to urobilinogen and excreted as a constituent of urine. In the various pathological conditions, conjugated bilirubin is itself excreted in the urine.
2. BRIEF DESCRIPTION OF THE PRIOR ART
Bilirubin is conventionally determined in routine urinalysis based on its reaction with various diazonium compounds in an acidic medium to form a colored azobilirubin complex. While several test formats are reported in the literature, the most commonly used test format in the clinical laboratory is that generally referred to as a test strip. The diazonium compound is incorporated in a carrier capable of absorbing a predetermined amount of urine when dipped momentarily into a urine sample. Any resulting colorimetric response may be read in less than one minute. The preparation and use of a bilirubin test strip is described in detail in U.S. Pat. No. 3,585,001. While the test strips which have been described in the art provide very rapid and convenient means for detecting urinary bilirubin, it is generally known that the available test strips are not sufficiently sensitive to detect levels of bilirubin only slightly elevated from the normal level, i.e. between 0.05 and 0.8 mg bilirubin per 100 ml.
There have been a few reported attempts to increase the sensitivity of the reaction between diazonium compounds and urinary bilirubin; however, the test systems that have resulted have certain disadvantages.
U.S. Pat. No. 3,880,588 describes a class of diazonium compounds designed to enhance the colorimetric response of the azobilirubin complex and to decrease interfering color reactions with urobilinogen, which is structurally and chemically very similar to bilirubin. The described diazonium compounds, unlike the conventional compounds, form interfering colored products with such constituents of urine as homogentisic acid and 5-hydroxyindole-3-acetic acid. The latter is a normal constituent of urine and as little as 1 mg/100 ml of such constituent in urine causes false positive results using the diazonium compounds described in this patent.
Another attempt to increase the sensitivity of the test strip-incorporated diazonium reagents is described in U.S. Pat. No. 3,853,476 which discloses the use of certain phosphoric acid diesters as sensitizing or potentiating agents for the reaction between the diazonium compound and bilirubin. However, due to the incompatibility between the phosphoric acid diesters and aqueous media, test strips prepared according to this patent must be manufactured by a double-impregnation process.
It should be mentioned that various so-called "accelerating agents" have been described in the art relative to the detection of bilirubin in serum by the diazo-coupling reaction. Such agents have included caffeine, dyphylline, sodium acetate, sodium benzoate, gum arabic, and various other chemically unrelated compounds. The use of such accelerating agents in serum bilirubin tests was described in the literature as early as the 1920's but has never been applied in general to urinary bilirubin tests. This has been due to the generally recognized fact that such accelerating agents act on a form of bilirubin that is not present in significant amounts in urine. Such accelerators are reported to promote the diazo-coupling of free bilirubin. No effect on the coupling of conjugated bilirubin has been reported, since in serum bilirubin tests the conjugated forms of bilirubin react relatively rapidly with the diazonium compounds without the need for accelerators. Hence, the conjugated forms of bilirubin in serum are referred to as direct-reacting bilirubin, whereas free bilirubin, which requires the presence of an accelerator in order to react rapidly, is referred to as indirect-reacting bilirubin. The fact that the scientific community views the effect of the reported accelerating agents as being restricted to the diazo-coupling reaction of free bilirubin, and not applicable to the reaction with conjugated bilirubin, is well supported by the concurrence of the primary review publications considered authorative in the art. For example, reference may be made to Henry, R. J., Clinical Chemistry, Principles and Technics, Harper and Row (1964) pp. 577-583; With. T. K., Bile Pigments, Academic Press (1968) pp. 324-327; and the Journal of American Medical Technologists, volume 31 (1969) pp. 707-710.
A single investigator has reported the use of dyphylline in a urinary bilirubin determination -- Scandanavian Journal of Clinical Laboratory Investigation, supplement 56 (1961). However, such use was specifically designed to accomplish the same effect as discussed in the literature relative to serum bilirubin tests, namely, to accelerate the diazo-coupling of free bilirubin which, as is known, could only be present in the urine tested in very small amounts. The described procedure involves a cumbersome liquid test system, and there is no suggestion of a rapid and sensitive test strip system. Moreover, the described procedure has received little attention from those skilled in the art in their development of more sensitive bilirubin test strips, as is evidenced by their resort to the disadvantageous test systems disclosed in the previously discussed U.S. Pat. Nos. 3,853,476 and 3,880,588.